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Master’s Thesis 2017 30 ECTS The Department of Animal and Aquacultural Sciences
Norwegian University of Life Sciences, Ås
Starch Digestion in Broiler
Chickens: A Literature Study and
an In vitro Comparison with Pigs
Sanan Talibov
Feed Manufacturing Technology
ACKNOWLEDGMENTS
I would like to express my profound gratitude to my supervisor, Prof. Birger Svihus for his guidance,
constructive feedbacks, encouragement and for giving me the privilege of being one of his students.
Thank you for your invaluable advice that not only contributed to this thesis' completion but will further
guide me in my future career.
I am grateful to Liv Torunn Mydland for her counseling in my laboratory work preparation and very
many thanks to Frank Sundby, Nina Pedersen and Ragnhild Ånestad for their kind assistance during my
laboratory work.
I am sincerely thankful to my friend Khalid Itani, who despite being extremely busy with his PhD, has
always found time for countless discussions, for giving advice and sharing his knowledge and
experience.
Marieke, thank you for your love, support, and belief in me.
Last, but not least, I am indebted to my old friend Prof. Arne Oddvar Skjelvåg for showing endless
support, hospitality, and generosity during my stay at his house. I cannot thank you enough!
Ås, May 2015
Sanan Talibov
ABSTRACT
Starch is a quantitatively important source of energy in poultry diets. Despite high loads of starch in the
diet, poultry species can utilize a variety of starch sources very efficiently. Pancreatic α-amylase is the
major responsible enzyme for starch digestion in birds and mammals. Considering high starch digestion
capacity of broiler chickens, a hypothesis was made that a fixed certain amount of chicken pancreas is
more effective at degrading starch than the same amount of pancreas from pigs. To test this hypothesis,
amylase activity of the pancreatic tissues from 9 broiler chickens and 9 pigs was determined by in vitro
starch digestibility analysis. The method was adapted from the total starch determination analysis with
some modifications to the procedure. Homogenates of all pancreas tissues were prepared to be used as a
source of α-amylase during starch digestibility analysis. Sieved wheat flour was used as a starch substrate.
Results of the study showed that there is no systematic difference between the same certain amounts of
pig and poultry pancreases on their ability to digest starch. However, correction of the pancreas weights
for the body weights of the species revealed that on average chickens have twice larger (P<.01) pancreases
than pigs relative to their body weights. Specific amylase activities (mg starch digested/min/mg protein)
were also not significantly different between species. Large individual variation was observed in enzyme
activities within both species.
Furthermore, enzyme kinetics of the starch digestion was also analyzed and discussed. The literature on
enzyme related factors of starch digestion was extensively reviewed and presented in this thesis.
Keywords: starch digestion, pancreas, amylase, broiler chicken, pig, enzyme activity
CONTENTS
1. Introduction .......................................................................................................................................... 1
2. Literature review .................................................................................................................................. 4
2.1. Distinctiveness of the digestive system of poultry ....................................................................... 4
2.2. Starch structure, characteristics, and digestibility ........................................................................ 6
2.3. Action of α-amylase and amyloglucosidase on starch .................................................................. 7
2.5. Enzyme related factors of starch digestion in chickens ................................................................ 9
2.6. Evaluation of analytical parameters for measuring α-amylase activity ...................................... 12
Summary ............................................................................................................................................ 13
3. Materials and methods ....................................................................................................................... 14
3.1. Background information on dissected animals ........................................................................... 14
3.2. Collection and storage of pancreases .......................................................................................... 15
3.3. Materials ..................................................................................................................................... 15
3.4. Pancreatic homogenate preparation ............................................................................................ 15
3.5. Determination of α-amylase activity .......................................................................................... 17
3.6. Enzyme kinetics .......................................................................................................................... 22
3.7. Data analysis ............................................................................................................................... 23
4. Results ................................................................................................................................................ 24
4.1 Kinetics of starch digestion.......................................................................................................... 25
4.2 Amylase activity of the pancreases .............................................................................................. 26
4.3 Protein analysis ............................................................................................................................ 27
4.4 Amylolytic activity ...................................................................................................................... 28
5. Discussion .......................................................................................................................................... 30
5.1 Kinetics of starch digestion.......................................................................................................... 30
5.2 Amylase activity of the pancreases .............................................................................................. 33
5.3 Protein analysis ............................................................................................................................ 37
5.4 Specific activity of α-amylase ..................................................................................................... 38
6. Conclusion ......................................................................................................................................... 42
References .............................................................................................................................................. 43
Appendix A ............................................................................................................................................ 50
Appendix B ............................................................................................................................................ 53
Appendix C ............................................................................................................................................ 56
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1. INTRODUCTION
Today’s modern poultry breeds exhibit extreme production performance compared to the ones in 1950s.
For instance, in 1956, a broiler breed needed 84 days to reach 1,82 kg, 10 years later this period was
shortened to 60 days, and as a result of intensive selection, in 2000 it took a broiler 34 days to reach the
same weight (Hafez and Hauck, 2005 as cited in (Buzała et al., 2015)).
Rapid growth of broiler chickens requires adequate amounts of energy to be supplied through the diet
alongside with protein, particularly essential amino acids. Most of the energy needed for fast growth of
broilers comes from starch which is quantitatively the major nutrient in broiler diets. It provides around
60% of apparent metabolizable energy (AME) content of poultry feed (Cowieson, 2005) and broiler diets
may comprise up to 50% of starch on dry matter (DM) basis (Svihus, 2014b). Several studies (Krogdahl,
1985, Krogdahl and Sell, 1989, Noy and Sklan, 1995, Wiseman et al., 1998, Tancharoenrat et al., 2013)
reported physiological limitations for lipid digestion in young birds. Impeded fat digestion was related to
either low pancreatic lipase availability or limited bile secretion by previously mentioned authors.
Although fats and oils possess about twice higher energy density (37 kJ/g) for the same mass of most
carbohydrates (17 kJ/g), yet, starch remains the primary source of energy for poultry.
Starch is a reserve carbohydrate that is present in many plants. The main sources of starch in commercial
broilers are cereal grains such as wheat (starch content1 ~70% DM), maize (~65% DM) and barley (~60%
DM). In cereal grains, starch is stored inside the granules in the endosperm. The granules consist of semi-
crystalline and amorphous layers and exist in various sizes and shapes. Amylose and amylopectin are the
main building components of layers and both are polymers of glucose units (Svihus et al., 2005).
α-amylase is the major responsible enzyme for starch digestion in birds and mammals. Although, there is
some α-amylase activity present in the saliva of humans and pigs (McDonald, 2002), in poultry, saliva
does not contain any α-amylase and pancreatic α-amylase is the only starch degrading enzyme (Moran,
1982, Wiseman, 2006).
There are several factors affecting starch digestion in poultry. Those factors can be grouped in two
categories: feed related factors: such as feed processing, particle size, starch/granule characteristics in the
feed, presence of fibers, antinutritional factors (ANFs), etc. and non-feed related factors: bird’s genetics,
age. Recent review by Zaefarian et al. (2015) explicitly discusses those factors causing variation in starch
1 http://www.feedipedia.org
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digestibility in broiler chickens fed cereal diets. In fact, pancreatic α-amylase activity does not take place
among factors affecting starch digestibility. Moran (1982) postulated that pancreatic α-amylase secretion
is never insufficient to support starch digestion in fowl and fowl is capable to increase the amount of
pancreatic amylase secretion to keep up with high levels of starch intake (Moran (1985) as cited in
(Zaefarian et al., 2015)). This statement was later supported by Noy and Sklan (1995) where they
quantified the amount of pancreatic enzymes secreted into the duodenum from 4 days to 21 day post-hatch
and revealed that amylase secretion increased more compared to the other enzymes. However, according
to the study amylase and protease synthesis during the first days of post-hatch can be limited. Gracia et
al. (2003) observed significant increase in starch digestibility when α-amylase was supplemented to corn-
soybean meal diets of broilers, hinting that endogenous α-amylase can be limiting for optimal starch
digestion. Kaczmarek et al. (2014) investigated the effect of amylase supplementation of starch
digestibility and growth performance of broiler chicks fed corn-soybean diet. Contrary to the earlier
studies no effect of amylase supplementation on starch digestibility and growth performance was observed
compared to the control (no enzyme) group. Authors recurrently concluded that digestive enzyme
deficiency in young chickens may not be as evident as thought. Generally, poor starch digestibility in
broilers is not attributed to inadequate α-amylase concentration in the small intestine (Wiseman, 2006).
Looking at starch digestion as a simple hydrolysis reaction, then another key factor would be time required
for complete hydrolysis. Too short exposure time to α-amylase may be one of the reasons for suboptimal
starch digestion (Svihus, 2011b). Feed intake level and particle size of the diet have shown considerable
impact on feed retention time in the digestive tract and hence on the starch digestibility. Svihus (2011b)
clearly illustrated declining total tract starch digestibility for increasing feed consumption of broiler
chickens receiving wheat based diet. This result was previously supported by Svihus (2006) where he
observed decreased AME values (due to lower starch digestibility) for broilers overconsuming pelleted
wheat-based diet. High levels of undigested starch in the excreta was detected by Péron et al. (2005) where
they provided finely ground wheat diet to broiler chickens immediately after food-deprivation, again
relating reduced starch digestibility to feed overload and consequently fast transit of feed through the
digestive tract. Feeding with whole cereals was also believed to decrease passage rate of feed through
upper digestive tract, however this hypothesis was not proved by Svihus et al. (2002), although Svihus
and Hetland (2001), Hetland et al. (2002), Svihus et al. (2004) observed improved starch digestibility
when broilers were fed unground cereals. Some of those aforementioned aspects of starch digestion in
chickens will be extensively discussed later with more emphasis on enzyme related factors.
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Pancreas is known to contain several enzymes as well as amylase (Berdutina et al., 2000). Comparative
study of pancreatic amylase activities between red jungle fowl and a broiler breed has been done by
Kadhim et al. (2011). Although significant differences were found in the enzyme activities between two
breeds, these differences were generally associated with differences in body weight. However, no
literature was found comparing pancreatic amylase activity of broilers with the activity of amylase from
pig pancreas. The aim of this study, was to compare the efficiency of chicken and pig pancreatic amylases
at degrading starch. Considering, very high starch digestion capacity of broilers a hypothesis was made
that a fixed certain amount of chicken pancreas is more effective at degrading starch than the same amount
of pancreas from pigs. To test this hypothesis, amylase activity of the collected chicken and pig pancreatic
tissues were determined by in vitro starch digestibility analysis in our lab. The literature on enzyme
activity, and other related factors of starch digestion were also reviewed and presented herein.
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2. LITERATURE REVIEW
2.1. Distinctiveness of the digestive system of poultry (chicken example)
Chickens are commonly known as granivorous poultry species. Although, Klasing classifies chickens as
omnivorous (Klasing, 1999, Klasing, 2005), it is a fact that cereal grains make up most of the poultry diets
and poultry possesses distinctive digestive anatomy to efficiently utilize even whole seed grains.
Unlike pigs and some other monogastrics, chickens cannot reduce feed size in the mouth. Ingested feed
moves down to crop which serves mainly as a storage organ in birds. Considerable moisturization takes
place in the crop, which may aid grinding and enzymatic digestion further down the digestive tract
(Svihus, 2014a). Crop is not thought to have a direct significant contribution to enzymic digestion, since
no enzyme secreted by the crop wall (Svihus, 2014a). However, Bolton (1965) investigated the digestion
process in the crop and reported extensive hydrolysis of starch (more than 20 %) into sugars in the crop.
Thereafter, Champ et al. (1983) identified three Lactobacillus strains from isolated chicken crop that
produce amylase, hinting to the fact that some microbial pre-digestion occurs in the crop. According to
Bolton (1965) some absorption of sugars and microbial fermentation of sugars to lactic and other acids
also takes place in the crop. Fermentation of simple sugars into acids by crop Lactobacillus species have
been well established (Sarra et al., 1992, Hilmi et al., 2007, Classen et al., 2016). In addition, it must be
noted that crop has a noticeable storage function under the situations of intermittent feeding, hence
transient store of the feed in the crop is not always the case for poultry which has ample access to feed
throughout a day (Svihus, 2014a).
Distinct to the pigs, chickens’ gastric region consists of two compartments called proventriculus
(glandular stomach) and ventriculus or gizzard (muscular stomach) (Klasing, 1999). Enzymic digestion
initiates when the feed meets proventriculus. The walls of the proventriculus secrete gastric juices:
Hydrochloric acid (HCl), pepsinogen (precursor of pepsin) and mucus. HCl and pepsinogen secreted by
the gastric glands initiates digestion of proteins by the action of later formed pepsin, and mucus secreted
by the tubular glands (Klasing, 1999) forms a protective coat to prevent gastric wall from the damage of
HCl and pepsin (Duke, 1994). However, due to the small volume of the proventriculus, retention time of
the feed is very short here and thus, gizzard is the main site where initial digestion takes place by the
action of gastric juices (Svihus, 2011a).
Gizzard has strongly myolinated muscles and a koilin layer, which aids in the grinding process due to its
sand-paper-like surface (Svihus, 2014a). Particle size reduction occurs when the feed leaves
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proventriculus and enters gizzard. Thus, the main function of the gizzard is grinding and mixing of the
feed which first action increases the surface area of the feed and the latter facilitates the action of digestive
fluids on feed particles. Refluxes from gizzard to proventriculus allows additional secretions to be added
to the feed (Duke, 1992 as cited in (Svihus, 2011a)).
Total tract retention time is lower in chickens compared with pigs, nevertheless, nutrient digestibility is
quite comparable with that of pigs (Moran and Practice, 1982). Average retention time in the stomach
region of chickens varies between half an hour and hour. Svihus et al. (2002) observed that significant
amounts of feed left gizzard in 30 min of feeding. Moreover, selective retention in the gizzard results very
short retention time of fine particles compared to coarse ones (Svihus, 2011a). When expressed as a
percentage of total tract retention time, the gastric retention time is quite similar between pigs and poultry
(Moran and Practice, 1982).
The gastric juice secreted by the proventriculus has been stated to have pH value around 2. However,
depending on the chemical characteristics of the feed, the pH in the proventriculus and gizzard of broiler
chickens has been reported to have an average value of 3-4 for normal pelleted diets (Svihus, 2011a). This
is consistent with the results observed by Jiménez-Moreno et al. (2009) where broilers were fed raw or
heat treated corn or rice with inclusion of some fibers. As feed passes to the duodenum pH rises to higher
levels above 6 (Svihus, 2014a) due to bicarbonate secretion from the pancreas (Fuller, 1991).
Small intestine is the site where enzymatic starch digestion initiates in the chicken. Total length of the
small intestine of the bird (1-2 m) is quite a bit shorter than in pigs (15-20 m). Also, addition of moisture
to intestinal chyme is less in chickens compared with pigs, resulting in a higher dry matter content of the
intestinal chyme (Moran and Practice, 1982).
Luminal cavity of the jejunum is the major site for starch digestion, since pancreatic duct opens up in the
beginning of the jejunum (Osman, 1982). Pancreatic α-amylase is the only enzyme responsible for starch
digestion in the fowl (Moran, 1982) since, chicken saliva does not contain α-amylase activity (Jerrett and
Goodge, 1973, Benkel et al., 1997). Although saliva of the pigs contains α-amylase, its activity is
negligible (McDonald, 2002). Hence, in monogastric animals, the major digestion of starch initiates in the
small intestine by the action of pancreatic α-amylase, and then followed by brush border enzymes
dextrinase and glucoamylase (Svihus et al., 2005).
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2.2. Starch structure, characteristics, and digestibility
Starch is the most abundant component in the diet of the poultry and other domestic animals. Apart from
animal related factors of starch digestion, digestibility of different native starches is closely related to their
structural and chemical characteristics. Therefore, it is crucial to have a basic knowledge about their
structure and composition.
Naturally starch is stored inside the granules which size and shape differ among plants. In cereals, starch
granules are accumulated in the endosperm. Granules consist of several layers which are composed of two
types of α-glucan: amylose and amylopectin (Buléon et al., 1998). They together represent approximately
98–99% of the dry weight (Tester et al., 2004). Both are the polymers of glucose units bound together
with glycosidic bonds. Amylose is made of α-D-glucose units bound to each other with α (1→4)
glycosidic bonds (99%). Therefore, amylose has a linear structure compared to amylopectin which is
highly branched. Besides α (1→4) linkages (95%) of glucose units in amylopectin, α (1→6) glycosidic
bonds (5%) (Tester et al., 2004) occur every 20-25 glucose units (Sajilata et al., 2006). This causes
amylopectin to get highly branched structure.
The proportion of amylose and amylopectin fraction depends on the source of the starch. Normally
amylopectin makes up to 70% and amylose 20-30% of starch (Svihus et al., 2005). Tightly packed
structure of amylose limits the accessibility of the digestive enzymes in ungelatinized starches with high
amylose content (e.g raw potato, banana) (Sajilata et al., 2006). High degree of crystallinity (high amylose
content) is associated with lower catalytic efficiencies and hydrolysis rate (Tahir et al., 2010). Moreover,
starches with high amylose content are also associated with high amounts of lipid formation (such as fatty
acids) on the surface of granules which can reduce the rate of enzymatic digestion (Svihus et al., 2005).
This will be discussed below in more detail. Amylose content may considerably differ depending on the
type and variety of cereals. In a wheat, amylose has been reported to vary within a range of 30-310 g/kg
which is smaller than barley (30-460 g/kg) and maize (0-700 g/kg). This smaller range for wheat is due to
the polyploid genomes which buffer variation in amylose content (as stated in (Svihus et al., 2005)).
Amylopectin ratio in wheat was reported to be 36 % (Morsi and Sterling 1966, Morrison et.al 1994) and
39 % (Yusuph et.al 2003) as stated in (Tester et al., 2004)).
Starch granule size can be another factor affecting digestibility of native starches. This is due to the
relationship between surface area and starch volume, which affects contact between starch substrate and
enzyme, that decreases as the granule size increases (Svihus et al., 2005, Dona et al., 2010). The granules
may vary in size from 1 to 50 μm. Cereals with smaller granules e.g rice 8 μm, have higher starch
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digestibility than the cereals with larger granules e.g wheat (22 μm) and potato (38 μm) (as stated in
(Svihus et al., 2005)).
There are some other non-starch components such as lipids and proteins in the starch granules that have
potential to interfere with starch digestion. For instance, lipid-starch complexes can reduce the access of
enzymes to starch. Lipids may further hamper digestibility due to their hydrophobic nature, thus reducing
the water access to granules and impeding the extent of gelatinization (Svihus et al., 2005). Other than
lipids, proteins found on the surface of granules (friabilin) may impair starch digestibility through
interactions during milling (Svihus et al., 2005). Matrix protein of the endosperm (gluten) may also
contribute to low digestibility of wheat starch (Wiseman, 2006, Svihus, 2011b, Svihus, 2014b).
The granular architecture and, more specifically, the surface organization of starch granules provide
barriers to the diffusion and adsorption of the enzymes, which is proposed to be one of the main actors
determining the kinetics and degree of hydrolysis (Slaughter et al., 2001, Tahir et al., 2010, Zhang et al.,
2013, Zhang et al., 2015, Dhital et al., 2017).
2.3. Action of α-amylase and amyloglucosidase on starch
The term “amylase” is generally defined as the enzyme which hydrolyzes the O-glycosyl linkage of starch
(Kuriki and Imanaka, 1999). α-amylase belongs to the endoamylase group of starch converting enzymes.
Endoamylases cleave α, 1-4 glycosidic bonds in the inner part of the amylose and amylopectin chains.
The breakdown products of α-amylase are oligosaccharides (e.g. maltose and maltotriose) and limit
dextrins with varying length (Van Der Maarel et al., 2002). However, there is also a possibility of glucose
appearence in the digesta due to action of pancreatic α-amylase. In an extensive study of the metabolism
of short-chain oligosaccharides by use of porcine pancreatic α-amylase, (Robyt and French, 1970)
observed that 70% of maltotetraose was cleaved to two molecules of maltose and 30% was cleaved to
glucose and maltotriose (as cited in (Kaufman and Tietz, 1980).
Using scanning electron microscopy technique Lynn and Cochrane (1997) observed wheat starch
digestion by the action of pancreatic α-amylase. According to their observations digestion initiated
through the channels on the surface of the central disc of the lenticular wheat starch granule and from
those channels digestion extended towards the interior of the granule before breaking apart the surface of
the granule (as stated in (Svihus et al., 2005)). Hydrolysis proceeds very rapidly in a radial direction with
the formation of new channels. Granular starch digestion occurs by a ‘side-by-side’ mechanism involving
the simultaneous digestion of crystalline and amorphous regions (Zhang et al., 2006), although the
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nonordered structure has been generally thought to be more easily digested (Gallant et al., 1992) as stated
in (Zhang et al., 2013)).
Amyloglucosidase was thought to act primarily on α-amylase breakdown products (Kaufman and Tietz,
1980), rapidly converting them to glucose. However, lately several authors (Miao et al., 2011, Brewer et
al., 2012, Zhang et al., 2013, Warren et al., 2015) have indicated synergistic action of α-amylase and
amyloglucosidase on attacking starch granules. As amyloglucosidase is also capable of hydrolyzing α-
(1→6) linkages, which α-amylase cannot. As in in-vivo digestion brush border enzymes maltase-
glucoamylase and sucrase-isomaltase can commit the same function (Nichols et al., 2003, Diaz-
Sotomayor et al., 2013) as cited in (Warren et al., 2015). This direct attack mechanism of
amyloglucosidase on starch granules was more evident in in-vitro digestibility trials of starchy foods with
high amylopectin content. Presumably, this was due to more branched structure of amylopectin containing
many α-(1→6) linkages.
This has important impact on interpretation of the results of in-vitro digestibility studies, as different
concentrations of enzymes may lead to doubtful estimations of the rate and extent of starch digestion
(Warren et al., 2015). Considering this fact, starch digestibility assay in our experiment was divided into
two successive steps as, after digestion of the substrate with pancreatic homogenate unreacted starch was
removed from the digesta, so that amyloglucosidase could only act on amylase products.
2.4. Kinetics of enzymic starch digestion
Kinetics of starch digestion in enzymic systems principally depends on two main factors: the
physicochemical architecture and molecular characteristics of the starch granule (amylose to amylopectin
ratio, starch granule size, etc.) and hydration (gelatinization) level of starch which increases the
availability of starch chains to digestive enzymes, thus affecting rate of hydrolysis (Dona et al., 2010).
Enzymic digestion of native starch is a pseudo-first order kinetic process, which the initial digestion curve
is linear with a constant rate at earlier time points until the substrate is not significantly depleted (Warren
et al., 2015). As the reaction continues the rate shows decay curve (Warren et al., 2015, Dhital et al., 2017)
and the kinetics of the hydrolysis can be studied by fitting first-order reaction equation:
Ct = C∞ (1 – e-kt ) (1)
where Ct is the concentration of product at a given time (t), C∞ is the concentration of product at the end
of the reaction, and k is the digestibility rate constant. For ease of interpretation, Ct may be expressed as
9
the amount of starch digested as a percentage of the total starch content of sample, calculated assuming
that all polysaccharide is converted to maltose (Edwards et al., 2014).
2.5. Enzyme related factors of starch digestion in chickens
α-amylase isozymes. α-amylase is single chained enzyme synthesized in the pancreas and salivary glands
and it exist at least in two isoamylase forms (Rodeheaver and Wyatt, 1984b). Lehrner and Malacinski
(1975) discovered three electrophoretically distinct pancreatic α-amylase isozymes in chicken pancreatic
tissues. In their study, they showed amylase to exist in the highest concentration compared to other
proteins. The results of the purification analysis of amylase isozymes showed that amylase proteins
accounted for 20-30% of the extractable proteins, which is higher than the rabbit (5%) and the rat (10.9%).
During the structural analysis of amylase isozymes Lehrner and Malacinski (1975) observed post-
translational modifications of amylase isozymes in crude pancreatic homogenates which was suggested
to be temperature, buffer, and pH dependent. However, authors stated that amylolytic activity of those
modified amylases (nongenetic isozymes) remained unchanged.
Effect of genotype. Several authors concluded that the levels of digestive enzymes are mainly determined
by the genetic stock (Nitsan, 1989, Nitsan et al., 1991b, O’Sullivan et al., 1992). Gapusan et al. (1990)
investigated the functional properties of two chicken allozymes (designated as AmyF and AmyS) in-vitro.
Statistical analysis of the study indicates significant differences between two of them in terms of specific
activities, substrate specificities and inhibitor sensitivities. AmyS showed significant specific activity
(maltose units per microgram of amylase) than AmyF. Activity of both amylases were negatively affected
by the α-amylase inhibitors from wheat, however, AmyS was less sensitive to protein inhibitors from wheat
than AmyF. This observation suggests that wheat utilization efficiency may differ between amylase
isozymes, depending on their ratio in the medium. Morever, in the study both amylases utilized different
substrates in the following order: amylopectin > potato starch > dextrin > glycogen > amylose. Since
amylose has the tendency to form helical coils of glucose when suspended in water (Conn et. Al 1987 as
stated in (Gapusan et al., 1990)) hence becomes more resistant to action of amylase. However, AmyF was
better able to attack α, 1-4 linkages of amylose than AmyS.
Yardley et al. (1988) examined amylase activity variation in three chicken flocks by sex, genotype (AmyS,
AmyF) and tissues. Significant differences in mean pancreatic amylase activities (maltose units per μg
protein) were found among three flocks. Variation among individuals within a flock is also reported in
the paper. However, there were no significant effects of sex and genotype on amylase activity. Highest
10
amylase activity was observed in the pancreatic tissue, the lower activity in the intestine and the lowest
activity was observed in the serum.
Effect of feed intake. Rodeheaver and Wyatt (1984a) studied the effect of feed intake on serum and
pancreatic α-amylase activities (units × 102 per g wet weight) in broiler chickens. Pancreatic α-amylase
levels elevated almost twofold in 20-day old male broiler chicks that were deprived from feed for 24
hours. The results of the study showes that pancreatic enzyme activity (concentration) was not reduced by
long-term feed restriction but it increased instead. Feeding for 24 hours following feed restriction caused
a sixfold reduction (from 440 to 77) in pancreatic α-amylase activity which was less than one-half of the
control value. This is explained as the compensatory effect to starvation and high need for glucose, thus
feed intake causes excessive release of α-amylase from the pancreas compared to the normal conditions,
which results in low levels of α-amylase in the pancreas itself. It is proposed that there is a certain basal
secretion of α-amylase from the chicken pancreas independent on feed intake. During feeding periods, α-
amylase secretion increases from the pancreas, however due to the release of the enzymes to the intestine
the level of α-amylase in the pancreas decreases. Without feed stimulation of the pancreas activity,
secreted α-amylase is accumulated in the pancreas, which results in increased levels of α-amylase in the
pancreas. This type of reverse relationship between amylase levels in the pancreas and intestines of
chickens was also noticed by Osman and Tanios (1983). Contrary to these findings, (Kadhim et al. (2011),
Kadhim et al. (2014)) did not find any correlation between the reduction of enzyme activities in the
intestinal content and any accumulation of these enzymes in the pancreatic tissue of the red jungle fowl,
Malaysian village chicken and the commercial broiler breed.
Nitsan et al. (1974) investigated enzymatic adaptability of young chicks in digesting and metabolizing
excessive amounts of nutrients under force-feeding and subsequent fasting conditions. Pancreatic lipase,
together with lipase and chymotrypsin significantly increased in the force-fed groups compared to the ad-
lib-fed group. Amylase activity (expressed as units/g) also increased in the small intestine contents of the
force-fed group, whilst other enzyme activities remained the same when expressed as units/g. However,
the specific activities of enzymes per g of tissue or chyme were the same in both groups despite the bigger
pancreas and high amount of feed in the intestines of the force-fed group. Similar results were observed
by O’Sullivan et al. (1992) where chicks from high-weight lines had elevated overall enzyme levels after
a mild feed restriction compared with those provided ad libitum access to feed.
Kokue and Hayama (1972) studied exocrine pancreatic secretion in starved chickens and observed that
the chicken is able to secrete the same volume of pancreatic juice after 72 hr starvation period as that of
11
the chicken starved for 24 hours. Feeding and sham feeding resulted in rapid increase in the volume of
secretions of the starved chicken. However, immediate increase was not observed in the vagotomized
chicken, although small delayed increase was noted. It is concluded that there is continuous synthesis (not
release) of pancreatic juice regardless of extraneous control mechanisms. Control by the parasympathetic
nervous system is suggested to be the reason for continuous pancreatic secretions.
Regulation. Murai et al. (2000) investigated the effects of different neurotransmitters and gut hormones
on pancreatic amylase secretion response in pancreatic acini in vitro. The researchers postulated that,
neural regulation of pancreatic enzyme secretion is more important than the humoral regulation. Based on
their findings cholecystokinin (CCK) at physiological concentrations did not influence amylase release
from isolated pancreatic acini of the chicken. This is in accordance with the findings of Niess et al. (1972)
where they did not observe significant effect CCK regulation on the pancreatic amylase secretion when
chickens were fed heated and unheated soybean proteins.
Effect of age. The level of α-amylase activity in the pancreas increases with the age of the bird. Rodeheaver
and Wyatt (1986) arrived at this conclusion where they examined pancreatic α-amylase activity by the
amylochrome method in different broilers at different ages. According to their observations, male broilers
at 7-weeks of age exhibited nearly 5 times increase in pancreatic α-amylase activity compared to the
activity of 11 days of age young chicks. At 20 days of age a moderate activity level of pancreatic α-
amylase was noticed. Progressive increase in the levels of activity is speculated to be the normal response
to increased metabolic needs of birds.
Similar tendency was monitored by Krogdahl and Sell (1989) where they measured different enzyme
activities in the pancreatic tissue in young turkeys and revealed that activity of amylase increased rapidly
during the first 14 days after hatch, whereas protease and lipase activities increased after 14 days. Opposite
to that Nitsan et al. (1991b) observed declining pancreatic amylase activity with increasing age in the
three different chicken populations. In the previous study by Nitsan et al. (1991a) the specific activity of
amylase did not change during the first 2 days but later increased continuously up to 17 days of age and
was 5-fold that at hatching.
Effect of diet composition. Several studies have shown that amylase activity of the birds can change with
diet as well as age. Hulan and Bird (1972) observed significant increase (P<0.001) in the amylase content
of the pancreatic juice per mg of protein when chickens received high amounts of carbohydrates. Krogdahl
and Sell (1989) monitored similar type of influence of feed composition to the levels of enzyme activity,
12
thus high carbohydrate and protein intake significantly increased amylase and protease activities of
pancreatic juice of young turkeys. Pubols (1991) reported the amylase to be the most abundant enzyme in
the chicken pancreas. The results of the ion-exchange chromatography showed that amylase was about
28.9%, chymotrypsinogens about 20% and trypsinogen was about 10% of total protein from the chicken
pancreata.
2.6. Evaluation of analytical parameters for measuring α-amylase activity
Such as other enzyme proteins, functional properties of α-amylase enzymes are also affected by extrinsic
factors such as pH, reaction temperature, storage temperature and duration prior to analysis, and presence
of certain ions for enzyme functioning. All these factors have significant contribution to the results of
enzyme assays thus, standardization of those parameters for assayed samples is necessary to keep
comparability of experimental results (Rodeheaver and Wyatt, 1984b).
pH and temperature. In general, α-amylases have relatively alkaline pH optima. Porcine pancreatic α-
amylase has the pH optima 6.9 for the majority of substrates (Ishikawa et al., 1991). Buonocore et al.
(1977) investigated the effect of pH on the activity of the chicken pancreatic amylase and reported that
enzyme activity stayed unaffected at alkaline pH values even after 24 h at 4 0C. But at pH values lower
than 7 activity noticeably decreased after 30 min. The effect of temperature on enzyme activity was tested
at the temperature range 20-60 0C. Maximum enzyme activity was observed at 43 0C at pH 7.5.
Storage temperature and duration. Rodeheaver and Wyatt (1984b) evaluated different analytical
techniques for quantification of α-amylase activity in serum samples. In the study poultry serum samples
were kept at different temperatures and durations and later samples were assayed for serum α-amylase
activity by three different (amylotube, amylochrome and Bernfeld 1955) techniques. Serum samples kept
at 40C showed large variation in α-amylase activity depending on the assay method used. The technique
of Bernfeld (1955) demonstrated significant decrease in activity after 24 hr and the lowest activity was
observed on the 4th day of storage. In contrary to that amylotube method showed consistent increase in α-
amylase activity through 8 day of storage at 40C. Serum α-amylase activities as measured by amylochrome
method did not significantly change during 8 day of storage at 40C. Freezing serum samples at -800C
significantly increased (by 32%) α-amylase activity compared to the fresh samples. It must be noted that
serum α-amylase is of pancreatic origin (Rodeheaver and Wyatt, 1986).
13
Rodeheaver and Wyatt (1986) observed more than 35-fold increase in the α-amylase activity (measured
by amylochrome method) when pancreas samples were sonicated after homogenization compared to the
samples that only followed homogenization. It confirms the intracellular nature of the pancreatic α-
amylase, indicating that the enzyme is secreted and stored in the zymogen granules of the pancreas and
subsequently released into the duodenum.
Solovyev and Gisbert (2016) tested the stability of digestive enzyme activities of gilthead sea bream stored
at -200C from 1 day up to 720 days. Up until 270 days, the change in α-amylase activity was still not
significant when samples were stored at -200C and authors recommended not to preserve samples at -200C
for digestive analysis more than 270 days.
Effect of ions. All amylases are believed to contain inorganic ions. Calcium has been found associated
with all amylases. Removal of this ion results in decreased enzyme activity which can be restored upon
re-addition of calcium ions (Vallee et al., 1959). In contrary, Buonocore et al. (1977) monitored
irreversible nature of the enzyme inactivation by the removal of calcium ions. It is generally believed that
calcium does not directly participate in th+e catalytic activity but it is involved in stabilizing the tertiary
structure (Buisson et al., 1987). Other than calcium, chloride ions also required for maximum enzyme
activity (Buonocore et al., 1977, Karn and Malacinski, 1978, Osman, 1982).
In-vitro analyses provide great advantages for speculation of in-vivo processes in animals. However, all
those aforementioned factors must be carefully considered prior to simulation of in-vivo starch digestion
to ensure the reliability of the results.
Summary
The pancreas has quantitively important role in starch digestion in pigs and poultry. Literature shows that
amylase in many species is continuously produced and stored in the pancreas until it is released. Several
factors such as age, diet, genotype, feed intake, etc. have shown to influence the level of α-amylase activity
in the pancreas. Moreover, synthesis and secretion of α-amylase appears to be differently regulated
between pigs and poultry. These aspects are important to take into consideration in the studies of pancreas
functionality.
14
3. MATERIALS AND METHODS
All laboratory work was conducted at three different labs at the Department of Animal and Aquaculture
Sciences of the Norwegian University of Life Sciences (NMBU) during the months of February and
March of 2017.
3.1. Background information on dissected animals
Chicken pancreases were obtained from an experimental trial that was conducted at the Animal Production
Experimental Centre, at NMBU, Ås, Norway, between the November 12th and December 4th 2015. The
experiment was a part of a comprehensive study, where the effect of different types of grit on broiler
chickens’ (Ross 308) performance was investigated. All birds had ad-libitum access to commercial
pelleted broiler diets from the Norwegian feed company Norgesfôr. The birds were fed starter diet from
day 0-11, grower diet from day 11-18. From 18-21 days of age birds got access to a mixed diet consisting
of 15% whole wheat and 85% protein-rich starter diet. 12 dissected chickens were selected from the birds
which 4 of them got zeolite, 4 marble and the rest for 4 got granite treatment. All birds were dissected at
21 day of age.
Pig pancreases were obtained from another experiment and belonged to the animals of the control group.
Pigs were fed with finisher diet and the main ingredient composition is given in table 1. According to the
information was obtained, animals were full-stomach before slaughtering. All pigs were dissected at 17
weeks of age.
Table 1. Main ingredient composition of broiler starter diet and pig finisher diet (%).
Broiler starter diet Pig finisher diet
Wheat 35.4 23
Maize 25.0 -
Barley - 40.9
Oat - 13.9
Soybean meal 23.3 13
Full-fat extruded soya 6.3 -
Rapeseeds 2 -
Rapeseed expeller 1.5 -
Lard - 4.8
Lysine 13.3 0.3
Methionine 6.3 0.6
Metabolizable energy (MJ/kg) 12.3 -
Net energy (MJ/kg) - 9.4
Crude protein 23.5 14
15
3.2. Collection and storage of pancreases
Dissection and collection of all pancreases were conducted by a PhD student Khaled Itani. All pancreases
were freed from surrounding fat tissue after dissection. After dissection pig pancreases were stored in a
freezer at -800 C for 12 months and chicken pancreases were stored in a freezer room at -200 C for 14-
month period until enzyme activity analysis. Only 9 pancreases from each species (18 in total) were
analyzed for α-amylase activity.
3.3. Materials
50 mM Tris-HCl buffer pH 8 and 0.2 M acetate buffer pH 6.1, (containing 200 mM CaCl2 and 0.5 mM
MgCl2) were prepared and kept in the fridge to be used during pancreatic homogenate preparation and
starch digestibility analysis. Fungal amyloglucosidase (Aspergillus niger) was obtained from Megazyme®
and had an activity of 3260 U/mL as defined by the manufacturer. One unit was defined by the
manufacturer as the amount of enzyme required to release one μmole of D-glucose reducing-sugar
equivalents per minute from soluble starch at pH 4.5 and 40oC.2 Ordinary sieved wheat flour was obtained
from the shop and used as a starch source in in-vitro digestibility analysis.
3.4. Pancreatic homogenate preparation
3.4.1 Sampling
The simplified sketch of pancreatic homogenate procedure is illustrated in figure 2. Each pancreas was
taken out from the freezer and weighed before preparation of a pancreatic homogenate. Immediately
after weighing, entire chicken pancreas was chopped into small fragments with a scalpel while pancreas
was still frozen. Pig pancreases possessed extremely huge mass and volume in contrast with chicken
pancreases. To avoid any possible methodical errors due to the great difference in the size of pancreases,
only representative subsamples were taken from pig pancreases. For this, each pig pancreas was divided
into half by a longitudinal cut (Figure 1a). Thin fragments were taken throughout the longitudinal cross-
section of a pancreas with the scalpel (Figure 1b). Fragments were then collected and weighed. Weight
of the subsamples were kept in the weight range of chicken pancreases.
2 https://secure.megazyme.com/Amyloglucosidase-Aspergillus-Niger
16
Figure 1. Sampling and preparation of pancreatic homogenates. a, b – sampling, c- homogenization.
3.4.2. Homogenization (enzyme extraction)
All pancreas samples followed the same subsequent steps during homogenate preparation. Chopped
fragments were placed into 15 ml propylene tubes and to this was added ice-cold (1:5 wt/vol) 50 mM
Tris-HCl buffer pH 8 (Pinheiro et al., 2004). The volume (ml) of the buffer was adjusted with the pipette
according to the weight (g) of a pancreas in order to keep the weight/volume ratio same for all pancreases.
See the example in Table 2.
Table 2. Buffer volumes corresponding to different pancreatic tissue weights. 1:5 wt/vol ratio. Pancreas
weight (g) 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0
Buffer
volume (ml) 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15
Propylene tubes were placed on ice and tissues were homogenized with “QIAGEN” TissueRuptor
handheld homogenizer for about 50 seconds until all tissue fragments disappeared (Figure 1c).
Homogenates were centrifuged at 4000 RPM 30 C for 12 minutes. Aliquots of supernatant were divided
into several small (1.5 ml) portions, (so that no repeated freezing and thawing of the same sample took
place throughout analysis) snap frozen in liquid nitrogen and stored at -800 C for later α-amylase activity
measurements and for total protein analysis.
b c
a
17
3.5. Determination of α-amylase activity
3.5.1 Principle
Determination of pancreatic α-amylase activity was based on in-vitro starch digestibility analysis with
pancreatic homogenates. Overall each digestibility assay can be divided into two main succesive steps.
The first step was starch digestion with pancreatic α-amylase. This step involves in-vitro starch
digestibility trials with pancreatic tissue homogenates. The procedure was adapted from the total starch
determination analysis by McCleary et al. (1997). However, as a major modification to the procedure,
pancreatic tissue homogenates were used (instead of thermostable α-amylase) to hydrolyze starch into
shorter oligomers. In the second step, those oligomers were further hydrolyzed into glucose monomers by
fungal amyloglucosidase. Glucose concentration of the final solution was measured in the
spectrophotometer. Glucose values were translated into starch and thus the percentage of digested starch
was calculated at different durations of starch hydrolysis reaction. Amylolytic activity of pancreases were
expressed as the amount (mg) of starch hydrolyzed per min per mg of protein.
3.5.2 Procedure
Each pancreas was tested for the enzyme activity in two parallels. For the sake of simplicity, the assay
procedure is described below as a single setup (see also figure 2).
Blanks. Parallel to the enzyme activity analysis “blank sample” analysis was run on every assay round.
All assay parameters were kept identical for blank samples, but no pancreatic tissue homogenate was used.
Instead of pancreatic tissue homogenate only homogenate buffer (Tris-HCl buffer pH 8) was added in 0.7
ml volume.
Starch digestibility analysis with a single pancreatic homogenate. Approximately 100 mg of wheat flour
was accurately weighed into 15 ml glass tube with a screw cap. 9 ml 0.2 M acetate buffer pH 6.1 containing
200 mM CaCl2 and 0.5 mM MgCl2 (Warren et al., 2015) was added into the tubes. A pancreatic tissue
homogenate was taken out from the freezer, thawed and was added into the tube in 0.7 ml accurate volume.
The mixture was incubated in water bath at 400 C for 150 minutes. Tube contents were mixed on a vortex
mixer every 10 min. Aliquots (1.2 ml) were taken at time intervals between 0 (before incubation) and 150
min (0, 15, 30, 60, 90, 120, 150) and centrifuged (3000 RPM for 5 min) to remove any unreacted starch
residue. Removal of unreacted starch at this step was important to avoid any possible amylolytic action
of amyloglucosidase (Warren et al., 2015) in the further step. Supernatant (1 ml) were then carefully
18
transferred to a new tube and immediately placed into boiling water to inactivate the action of pancreatic
α-amylase (Slaughter et al., 2001). From that 0.925 ml was added into 5 ml propylene tubes containing 3
ml 200 mM acetate buffer pH 4.5 (McCleary et al., 1997) and 0.075 ml fungal amyloglucosidase
(Megazyme®) mixture to degrade all amylase breakdown products to glucose. This was then incubated in
water bath at 500 C for 30 min. Tube contents were mixed on a vortex mixer every 10 min. Then tube was
placed into boiling water for 5 min to deactivate amyloglucosidase. After boiling, the tube was centrifuged
at 5300 RPM for 10 min to remove coagulated enzyme proteins from the solution. 300 μl sample was
taken from the supernatant and analyzed for glucose concentration in MaxMat spectrophotometer.
Measurement principle of the spectrophotometer is based on colorimetric glucose-hexokinase method.
(Richterich and Dauwalder, 1971). Required reagents for glucose measurements were obtained from
DIALAB®. Test principle of the method is given below as described by the manufacturer:
ATP + Glucose ℎ𝑒𝑥𝑜𝑘𝑖𝑛𝑎𝑠𝑒
→ Glucose-6- phosphate + ADP
Glucose-6- phosphate + NAD+ G6PDH
→ 6-Glucose phosphate ester + NADH + H+
Glucose in the sample, under the catalysis of hexokinase and glucose-6 - phosphate dehydrogenase
(G6PDH), react with ATP to produce glucose - 6 - phosphate and adenosine diphosphate. Glucose - 6 –
phosphoric acid is oxidized to 6 - phosphate glucose in ester, meanwhile, NAD+ in the reagent is reduced
to NADH, cause the increase of light absorbance value at 340 nm. NADH volume is proportional to the
amount of glucose. By measuring the change of absorbance value at 340 nm, the concentration of glucose
can be calculated:
Glucose concentration (mmol/L) = Absorbence value of sample tube
Absorbence value of calibration tube × Concentration of Calibrator (mmol/L)
Starch digestibility. Average of all blank values at each duration were subtracted from the glucose values
of starch digestibility analysis to obtain glucose concentration reflecting only starch hydrolysis by the
action of pancreatic α-amylase. In other words, since the assay procedure did not include removal of free-
sugars from the substrate, it was believed that pre-existing free-glucose in wheat flour would overestimate
starch digestibility.
19
Digestibility of starch was calculated according to the following formula:
Starch digestibility (%) =𝐺 × 180 × 0.9 × 0.001 × 𝑑𝑓 × 𝑉
𝑆 × 100 %
Where: G = glucometer reading (mmol/L), 180 = molecular weight of glucose, 0.9 – factor to convert
glucose concentration into starch, 0.001 = conversion factor from L to ml, df = dilution factor in the
digestion step with amyloglucosidase, V = volume (ml) of the digesta with pancreatic α-amylase, S = the
amount of starch added (mg) into the digesta.
Wheat flour from the shop was analyzed for the starch content in our lab and it was determined to contain
74.7 % starch on as is basis.
Relative weight of pancreases. Body weights of the animals before dissection were obtained from
previous experiments. Relative pancreas weights were estimated by expressing pancreas weights as a
percentage of animals’ body weights.
20
Fig
ure
2.
Sket
ch o
f th
e pan
crea
tic
ho
mo
gen
ate
pre
par
atio
n p
roce
dure
and s
tarc
h d
iges
tibil
ity a
ssay
.
21
3.6. Protein analysis
To determine the protein concentration of the pancreatic homogenate solutions protein analyses were
conducted by me at the Cigene lab of the university under the supervision of PhD student Ragnhild
Ånestad. Analyses were run according to the Quick StartTM Bradford protein assay (Bradford, 1976). After
the analysis known concentration of homogenate solutions were used to calculate the soluble protein
content of the pancreases.
3.6.1. Principle
The Bradford assay is a protein determination method that involves the binding of Coomassie Brilliant
Blue G-250 dye to proteins. The binding of the dye to protein causes a shift in the absorption maximum
of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored
(Bradford, 1976).
3.6.2. Selecting a protein standard
Bovine serum albumin (BSA) protein with a known protein concentration (2 mg/ml) were used to develop
a standard curve for the assay. Since pancreatic homogenate samples were made in Tris-HCl buffer pH 8,
the same buffer was used as a diluent (instead of water) throughout the assay. The compatibility of the
buffer with the assay was tested against double-distilled water. Despite the slight dissimilarity in the
curves it was believed that using Tris-HCl buffer as a diluent will not affect the comparability of the
samples (Appendix B fig. 1.)
3.6.3. Assay protocol
Analysis were conducted according to the microassay protocol3 in 300 μl microplates. The linear range of
this assay for BSA is 1.25-10 μg/ml, therefore, samples were diluted several times to find appropriate
protein concentration fitting to the linear range of a standard curve (Appendix B fig. 2). Final dilution
factor was determined at 1:3000 homogenate to buffer ratio. Standard and sample protein solution were
pipetted into the microplate wells in 150 μl volumes and to this was added 150 μl dye reagent (Coomassie
Brilliant Blue G-250) to get the color response. Microplate was then placed into spectrophotometer and
statistical means were obtained from a software (SoftMax). All protein solutions were assayed in
3 http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf
22
triplicates. All results (raw data) are presented in the Appendix B table 1. The mean values were multiplied
by the dilution factor to obtain the protein concentration of the pancreatic homogenates.
Average amylolytic activity. Average digestion rate (%/min) for each pancreas was first determined by
dividing the final digestibility values by the total reaction time (150 min). Percentages were converted
into mg of starch and values were corrected for the protein content of the pancreases. Average amylolytic
activity (specific activity) of the pancreases were expressed as mg of starch hydrolyzed per minute per
mg of protein (León et al., 2014).
3.6. Enzyme kinetics
The rate of starch digestion is very important for determining the glycemic responses of starchy foods for
humans. Based on this phenomenon Englyst et al. (1992) classified starch sources in three categories as:
rapidly digestible, slowly digestible and resistant starch. Thus, it is well established that in starch
containing foods digestion of the different starch fractions occurs at different rates (k). Poulsen et al.
(2003) developed a method called “Logarithm of Slope” (LOS) analysis which was later used by many to
examine those changes in reaction rates. The slope is sensitive to changes in k occurring during a reaction
and these changes are revealed by discontinuities in the linear plot (Butterworth et al., 2012). LOS analysis
enable scientists to determine the reaction rate constants (k) at divergent phases of reactions and true
reaction end points (C∞). Since a thorough understanding of starch digestion kinetics was not the focus of
this thesis, LOS plots were only obtained to reveal occurrence of distinct phases during starch digestion
in our experiment. A LOS plot was obtained by expressing the first derivative of the first-order equation
(see section 2.6 eq. 1) in logarithmic form:
𝑙𝑛 (∆𝐶
∆𝑡) = −𝑘𝑡 + ln (C∞ k) (2)
where ln(∆C/∆t ) represents the logarithm of the slope, and the equation describes a linear relationship
between LOS and time of amylolysis, t (as described by Edwards et al. (2014). Thus, a plot of natural
logarithms of (C2 – C1)/(t2 – t1), (C3 – C2)/(t3 – t2), etc. against t is linear with a slope of -k (Butterworth et
al., 2012). Because logarithms are plotted, C values do not have to be concentrations. They can be any
measured parameter that is directly related to concentration (Butterworth et al., 2012). Hence, starch
digestibility values (%) were employed instead of concentrations.
23
3.7. Data analysis
Statistical analysis of the data was performed in IBM SPSS Statistics 23 software. Starch digestibility
values were subjected to one-way ANOVA using General Linear Model (GLM) procedure. The variance
in relative pancreas weights of the species was analyzed by independent sample t tests. Simple linear
regression analysis was performed to examine the relationship between enzyme activity and protein
content of the pancreases. Mean values and standard deviations were calculated and illustrated by using
math and graphical functions of Microsoft Office Excel 365. Plots of the LOS (Butterworth et al., 2012,
Edwards et al., 2014) of the experimentally obtained digestibility curves were generated in Microsoft
Office Excel 365 to establish whether amylolysis followed a single-phase or two-phase hydrolysis
reaction.
24
4. RESULTS
The main objective of this thesis work was to test the hypothesis whether chicken α-amylase was more
effective at degrading starch than pig α-amylase for the same amount of pancreatic tissue. For this, we
conducted in-vitro wheat starch digestibility analysis with 9 pig and 9 chicken pancreatic homogenates
and the results of the analysis are presented below.
Figure 3. Starch digestibility curves at different times (○ pig, ● chicken, □ blank)
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120 140 160
Sta
rch d
iges
tib
ilit
y (
%)
Time (min)
25
4.1 Kinetics of starch digestion
Blanks. Results of the blank values at all digestibility trials are given in Appendix A fig. 1. As would be
expected, in the absence of α-amylase, the release of glucose from the wheat flour was nearly zero.
However, very low glucose (1-2 %) was detected in the blank samples where no pancreatic solution was
added. There was no significant (P>0.05) increase in glucose at increasing reaction times in the blank
samples. High variability in glucose values was noticed between measurements at all durations (CV %
ranging from 33 % to 95%, Appendix A fig.1)
Reaction kinetics at low and high enzyme concentrations. Digestibility analysis revealed large variability
in starch digestibility among individuals from both species. However, for both species during the first 30
minutes on average ̴ 22 % of starch was digested. From 30 to 60 min ̴ 12 %, 60-90 min ̴10 %, 90-120
min ̴ 8 %, and during the last half an hour ̴ 7 % of starch was hydrolyzed.
LOS of the highest (96 %) and the lowest (28 %) digestibility curves were plotted to observe enzyme
kinetics at two different enzyme concentrations. LOS plots revealed two distinct linear (R2 > 0.9) phases
for both reactions which can be characterized by two different rate constants (figure 4).
Figure 4. Logarithm of slope (LOS) plots of the highest (upper line) and the lowest (lower line) starch
digestibility curves. (I, III – rapid phases, II, IV – slow phases)
R² = 0.93I
R² = 0.91II
III
R² = 0.93IV
0.00
0.05
0.10
0.15
0.20
0.25
0 20 40 60 80 100 120 140
LOS
Time (min)
26
The point at which the slower phase becomes predominant is represented by the intersection between the
two linear phases. At high enzyme concentration, the slower phase initiates 30 minutes after the
commencement of incubation, whilst at low enzyme concentration this occurs earlier, as 10-15 min after
the onset of incubation. These results revealed that digestion of the wheat flour in our experiment followed
two: rapid and slow phases, independent of the enzyme concentration.
4.2 Amylase activity of the pancreases
As can be seen from the figure 3 both species showed large variation in the rate and extent of wheat starch
digestion. Besides large individual variation within the species remarkable variation was present between
the species. Variation among chickens was even greater than pigs (figure 5). The maximum and minimum
digestibility values were obtained from the digestion with chicken pancreases which were 96.8 % and
28.6 % respectively. The values for pig pancreases varied within the range from 31.7 % to 87.1 % of
starch digestibility. Average starch digestibility at the end of the incubation was 61 % for pigs and 60 %
for chickens (insignificant difference at α >0.05).
Figure 5. Average starch digestibility. Values are shown as means + S.D.
As expected, both species showed systematic increase (P<0.05) in starch digestion over time. However,
average starch digestibility at each time duration was not significantly different (P>0.05) between two
species (Figure 5). The results of the GLM procedure also did not show any significance (P>0.05) for the
effect of species on starch digestibility (see Appendix C, table 2) These results indicated that there is no
0
10
20
30
40
50
60
70
80
90
100
0 15 30 60 90 120 150
Sta
rch d
iges
tib
ilit
y (
%)
Time (min)
Pig Chicken
27
systematic difference between same certain amounts of pig and poultry pancreases on their ability to digest
starch.
Figure 6. Averages of the relative pancreas weights for both species. Values are shown as means + SD, P<0.01.
Correction of the pancreas weights for the body weights of the animals showed that average of the relative
pancreas weights for the pigs was 0.12 %, whilst for chicken this value was 0.24 % with relatively large
standard deviation (figure 6). Independent sample t test showed that the difference in the mean values was
significant at 0.01 significance level (Appendix C table 3).
4.3 Protein analysis
The results of the protein analysis are presented in the figure 7. From the plot, it can be seen that soluble
protein content of the pancreases showed considerable variation among individuals within the species.
This variation was even larger among chickens with values ranging from 6 % to 16%. For pig pancreases
values lied in between 11-14%. Simple linear regression analysis (Appendix C table 1) showed weak
correlation (r=0.4 for pig, r=0.1 for chicken) between soluble protein content of the pancreases and the
total starch digestibility. However, correlations were not statistically significant for both species.
Interestingly, the pancreas with the lowest protein content (6 %) exhibited the highest starch digestibility
(96 %).
0.00
0.05
0.10
0.15
0.20
0.25
0.30
REL
ATI
VE
PA
NC
REA
S W
EIG
HTS
(%
)
Pig
Chicken
28
Figure 7. Relationship between starch digestibility and soluble protein content of the pancreases (○ pig, ● chicken)
Rpig=0.4 for pig, Rchicken=0.1. Correlations are not statistically significant (P>0.05)
Overall protein data showed that soluble protein content of the pancreases was not a potent predictor of
α-amylase activity in our experiment.
It must be mentioned that extraordinarily low protein value of the pancreas of the chicken individual #8
is not thought to be due to any practical or methodical errors during analysis. Results displayed consistent
low values for that pancreas after several attempts in protein quantification analysis.
4.4 Amylolytic activity (Specific activity)
Once amylase activities were corrected for the protein content of the pancreases very diverse results were
obtained (figure 8). The chicken pancreas with the highest α-amylase activity (individual #8) had the
lowest protein content, hence correction for the protein content showed extremely high amylolytic activity
for that pancreas.
Average amylolytic activities (mg starch/min/ mg of protein) of the pancreases showed higher value
(0.021) for chickens compared to pigs (0.017). Although this difference was insignificant at α =0.05.
Obviously, higher average amylolytic activity value of the chickens was highly influenced by the
extremely high value of the chicken #8 (figure 9).
10
20
30
40
50
60
70
80
90
100
5.0 7.0 9.0 11.0 13.0 15.0 17.0
Star
ch d
iges
tib
ility
(%
)
Protein content (%)
29
Figure 8. Amylolytic activity as expressed mg starch digested/min/mg protein
Figure 9. Average amylolytic activity (mg starch digested/min/mg protein). Values are shown as means + S.D.
P>0.05
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
1 2 3 4 5 6 7 8 9
Am
ylo
lyti
c ac
tivi
ty
# individuals
Pig Chicken
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
AV
ERA
GE
AM
YLO
LYTI
C A
CTI
VIT
Y
Pig
Chicken
30
5. DISCUSSION
5.1 Kinetics of starch digestion
Although, the digestibility assay in this current study was not designed in a way to study the kinetics of
starch digestion, yet, the digestibility curves allowed to observe some patterns of enzymic starch
hydrolysis.
Blanks. Looking at the blank data, it would be hard to comprehend any possible action of intrinsic α-
amylase (Major et al., 2001) in the wheat flour. Since, there was no significant increase in glucose at
increasing reaction times in the blank samples. Moreover, large variation between replicates may also
indicate that measurements were highly sensitive to practical errors. However, compared to the samples
with pancreatic tissues, blank samples showed fairly steady low levels of glucose throughout the reaction
which is more attributable to the level of pre-existing free glucose in the wheat flour.
Reaction kinetics at low and high enzyme concentrations
First of all, it is more logical to think that varying digestibility values in our experiment is more likely to
be due to varying α-amylase concentration in the pancreatic tissues. Since the same starch source was
used and the assay parameters were kept identical throughout the experiment.
The reaction of α-amylase on soluble (gelatinized) starches is a single-phase first-order reaction
(Butterworth et al., 2012, Edwards et al., 2014) and can be described by traditional Michaelis-Menten
kinetics where, the rate of the reaction is directly proportional to the enzyme concentration. Thus, as the
reaction proceeds and the substrate is depleted the reaction rate shows an exponential decay curve
(Slaughter et al., 2001, Dhital et al., 2017). However, hydrolysis reaction of insoluble native
(ungelatinized) starches is rather complex due to their granular architecture. The shape of the curve
(logarithmic or linear) is affected by the level of enzyme activities. Butterworth et al. (2012) stated that
when starch or starch-containing foods are digested in-vitro with relatively high concentrations of
pancreatic amylase and for long time periods, i.e., extending over several hours, the rate of the reaction
decreases as the time is extended and plots of the concentration of product formed (or quantity of starch
digested) against time are logarithmic. Slaughter et al. (2001) investigated the action of porcine pancreatic
amylase on native starches and observed that the rate of amylolysis of native starch suspensions was
essentially linear for up to 2 h but the rate over the first 60 s or so was often higher than over the remaining
time course when digestion products were measured by reducing sugar assay. Independent of the enzyme
concentration, the similar pattern was also observed for the digestibility curves in our experiment. Thus,
31
computed linear lines to digestibility curves up to 2 hours provided a very good fit with R2 > 0.9 (the graph
is not shown in this thesis). However, declining digestibility values throughout sampling times makes it
clear that lines are not of true linear form.
The binding step of α-amylase to starch granules is of kinetic significance when the enzyme is acting on
particulate starch, for instance, α-amylase of Bacillus subtilis has been shown to adsorb to crystalline
starchy materials and the binding is a prerequisite for catalysis (Leloup et al. (1991) as cited in (Slaughter
et al., 2001)). Thus, for the enzymes acting on insoluble substrates the rate is not directly proportional to
the enzyme concentration (Slaughter et al., 2001) and usually reaction occurs in two phases: rapid and
slow phases.
Edwards et al. (2014) monitored a single-phase hydrolysis reaction for wheat flour. In contrary, the results
of the LOS analysis of the lowest and the highest digestibility curves revealed two distinct phases of wheat
starch digestion in our experiment. At high enzyme concentration, rapid hydrolysis phase lasted up to ̴30
minutes and followed by a slow phase for the rest of the reaction time. This was in accordance with the
findings of Butterworth et al. (2012) where they observed the rapid phase lasting 20-30 minutes during
in-vitro digestion of native wheat and pea starches. At low enzyme concentration, relatively short (10-15
min) rapid phase was observed and the onset of the slow phase shifted to earlier time points. Although
discontinuity occurred in both LOS plots, it was more evident for higher starch digestibility curve. Overall,
LOS plots showed that digestibility curves in our experiment were of discontinuous semi-logarithmic
form.
Despite the fact, that digestibility curves at relatively low enzyme concentrations seems to level out close
to the end of the reaction time (2.5 h), obviously, that decay in the digestion rate cannot be linked to
substrate depletion, since for most of the trials considerable amounts of starch remained undigested after
the end of the reaction. Surprisingly, even at high enzyme concentrations where almost more than 90% of
starch was digested, curves did not appear to plateau after 2.5 hours. Explanation for this remains a bit
unclear, since the reaction time was restricted to 150 minutes which considerably limits the amount of
information that may be obtained about the progress of the reaction.
Warren et al. (2015) postulated that at low enzyme activities the observed digestion curves are essentially
linear form, where insufficient substrate is converted to the product during the time course of the reaction
that results in significant decay in overall rate of reaction. According to the definition of a first-order
reaction, it is a reaction that proceeds at a rate that depends linearly on only one reactant concentration.
32
Hence, fitting starch hydrolysis to a first-order reaction would only be plausible when the amount of
enzyme in the system relative to the substrate is not a limiting factor for hydrolysis. Slaughter et al. (2001)
postulated that fraction of enzyme molecules bound productively to the starch granules is minor compared
with the total amount of enzyme in the system. Interpreting this in a different way, it could also be said
that at relatively high concentrations of α-amylase in the system, the rate of the reaction is primarily
dependent on starch concentration. The more the starch, the more productive binding and hydrolysis. This
has an important implication to the assay method that was used in our experiment. Thus, the removal of
considerable amount of digesta (>12 % of the total) at every sampling time (0, 15, …, 150 min) would
have also resulted in removal of substantial amount of starch from the system. As a result of this, it is
reasonable to speculate impairment in the hydrolysis rate throughout sampling procedure and in overall
rate of the digestion.
High-substrate inhibition. Inhibition of enzymic processes is normally either competitive, where the
inhibitor (in this case maltose) binds to the enzyme competitively with the substrate, or non-competitive,
in which case the inhibitor has identical affinities for the enzyme and the enzyme–substrate complex,
decreasing the maximum velocity of the reaction (Dona et al., 2010). Both types of inhibition of starch
hydrolysis can occur during in vitro enzymic digestion of starch. One of the special forms of
uncompetitive inhibition is where the inhibitor binds with enzyme-substrate complex and decreases the
reaction rate at high substrate concentrations (Dona et al., 2010). This type of inhibition mechanism will
not be further discussed here, since product inhibition of starch digestion appears to be more probable for
the digestion kinetics than substrate inhibition in our experiment.
Product inhibition. Oligosaccharides (e.g., maltose and maltotriose) are known to be competitive
inhibitors of a-amylase (Leloup et al., 1991, Elödi et al., 1972, Butterworth et al., 2012). During prolonged
periods of starch digestion maltose which is the main product of amylase digestion may accumulate and
inhibition of starch hydrolysis can become significant (Elödi et al., 1972). However, during the initial
stages of starch digestion, maltose levels in the digesta is not sufficient to have a significant inhibitory
effect on α-amylase action (Warren et al., 2015). Morever, it was hypothesized that under certain
conditions, especially at high concentrations of maltose, two maltose molecules can bind to the active site
of the porcine pancreatic α-amylase (Warren et al., 2012). In the study by Warren et al. (2012) where they
investigated maltose inhibition during the hydrolysis of maize starch by porcine pancreatic amylase and
concluded that, even when maltose concentration is sufficiently high, the affinity of the binding of the
second molecule to the active site is approximately 6.5-fold weaker than the first binding. Because of the
33
relatively low affinity for maltose, authors postulated that inhibition by maltose of the initial stage of
starch-amylase interaction normally does not play an important role in regulating intestinal digestion of
starch (Warren et al., 2012). Considering these findings, its logical to think of any potent inhibitory effect
of maltose on starch digestion during our experiment. Commonly, in in-vitro digestibility assays,
combination of α-amylase (or pancreatin, containing α-amylase) and fungal amyloglucosidase are used.
In such cases, amyloglucosidase rapidly converts all α-amylase products into glucose which has an
advantage of eliminating inhibitory effect of maltose accumulation during prolonged digestibility trials
(Warren et al., 2015). Digestion steps with α-amylase and amyloglucosidase were isolated in our
experiment. Since no combination of these enzymes was used, declining rate of digestion over prolonged
durations can partly be due to accumulation of α-amylase breakdown products and particularly maltose.
Enzyme activity analysis of pancreatic tissues by means of in-vitro digestibility assays offer great
advantages for the study of enzyme kinetics. However, comparability of the obtained data with literature
data is often challenged because of the differences in assay systems. Several aspects must be carefully
considered while establishing methods to achieve results that are broadly open for discussion.
5.2 Amylase activity of the pancreases
Generally, amylase activity of the pancreas refers to the level of α-amylase (concentration of α-amylase)
in the pancreatic tissue and should not be confused with the specific activity of the α-amylase where the
activity of the enzyme is corrected for the protein content of the enzyme containing solution.
Ultimate purpose of the current study was to test the starch digestion capacity of the same amount of
pancreatic tissue from chickens and pigs and the results of the starch digestibility analysis showed that
there is no significant difference (P>0.05) in starch digestion capacity between the two species. Because
of the exclusive design of the study it was challenging to find comparable data in the literature to explain
the reasons for large variation in amylase activity among individuals from both species. Extensive
literature review indicated that amylase activity is usually studied under specific conditions (e.g. exposure
to heat) or treatments (diet, age, genotype etc.). It must be mentioned that experiments which pancreases
were obtained, were not designed for pancreatic enzyme activity studies. Nevertheless, results can be
discussed assuming that the selected pigs and chickens for the present study were quite representative for
the population of pigs and poults in general.
34
Somewhat similar to our study Kadhim et al. (2011) investigated enzyme activity of the pancreas in two
breeds of chicken that differ in growth rate. Comparative data on enzyme activities (units/gram of
pancreas) showed that level of amylase in the pancreas of commercial broiler chicken (CBC- characterized
by high-growth rate) was significantly (P<0.05) higher than the red jungle fowl (RJF - characterized by
low-growth rate) at different ages from hatching to 120 days. However, when results were expressed in
relative basis as units/100 g of body weight no significant difference was detected after 20 days between
the two breeds. Similarly, absolute activities (U/g) of duodenal and jejunal amylase of CBC were
significantly higher than RJF at all ages, but when the results were expressed as U/100 g BW this
relationship was reversed, except for 1 day after hatching. In other words, data failed to follow the rapid
gain for body weight of CBC when amylase activity was corrected for body weight, except for the 1 day
post-hatching. Moreover, although absolute amylase activities (U/g pancreas) increased for increasing
age, however, relative amylase activities (U/100 g BW) showed increase up to 10 days post-hatch and
reached to the peak level and then declined with age for both strains. At 10 days post-hatch amylase
activity of CBC was approximately 3 times higher (p<0.05) than RJF. Authors, postulated that the body
requirements had important effect on the synthesis of enzymes, however major source of differences in
enzyme activities was due to the genetic stock of two chickens. Identical pattern of differences in absolute
and relative amylase activities was observed when Kadhim et al. (2014) studied differences in enzyme
activities in pancreas and intestinal contents of Malaysian village chicken (MVC - characterized by low-
growth rate) and CBC (high-growth rate). As in the previous study, CBC showed significantly higher
amylase activities per g of pancreatic tissue or intestinal content compared to MVC, but once the results
were corrected for body weight no significant difference in amylase activities was present between the
two breeds. These findings elucidate that high levels of amylase activity in the pancreas of broiler chickens
is paralleled by high growth rate which shows that synthesis of the pancreatic enzymes is adjusted
according to bird’s body requirements.
In order to reveal whether the similar relationship existed among birds and pigs in our study, the pancreas
weights were corrected for the body weight of the animals and thus relative pancreas weights were
obtained. Weights of the animals (appendix A table 2) did not vary to a large extent within both species
(since individuals from both species were killed at the same age) thus, it was not possible to observe
changes in the relative pancreas weights in intra-species level. However, results of the relative pancreas
weights between two species revealed that on average chickens had twice larger (P<.01) pancreases than
pigs relative to their body weights. This seems to be logical considering high growth rate of the broiler
chickens and thus relatively high body requirements for the enzymes. Moreover, maintenance energy
35
requirements for the same body weight of birds can be presumed to be relatively higher than pigs
depending on their body composition (ratio of fat to lean body tissue) and furthermore, considering the
fact that body temperature of the birds is relatively higher than pigs. These are just hypothesized subjective
opinions suggested to explain significant differences in relative pancreas weights of pigs and chickens.
Feed restriction. The reverse relationship between amylase levels in the pancreas and in the intestine, was
briefly discussed in the literature review part (see section 2.5). According to the information received,
dissected broiler chickens in the present study were deprived from feed at day 20 from 9 pm until 7 am
next day. On that day birds were given ad-libitum access to feed again until they were dissected at about
12 am. It can be speculated that during ̴4 hours birds would have normalized the secretion of pancreatic
enzymes. However, there are uncertainties regarding the rapidity in the response of pancreas secretions to
the feed intake in chickens. For instance, Pinheiro et al. (2004) studied the effect of 30% feed restriction
in broiler chickens from 7 to 14 days old chickens and revealed adaptation of digestive enzymes during
and after feed restriction. Thus, birds lowered the secretion of enzymes during the restriction period.
However, amylase, trypsin and lipase levels increased in feed restricted birds immediately after feed
restriction compared to non-feed restricted group. The increase is considered to be one of the factors
contributing to compensation in delayed growth due to energy restriction (Pinheiro et al., 2004).
Rodeheaver and Wyatt (1984a) on the other hand, observed a six-fold reduction in pancreatic amylase
activity after feeding for 24 hours following feed restriction (starvation) which was less than one-half of
the control value. Based on this record, it can be stated that even 24 hours of feeding subsequent to
starvation is not enough to return the pancreas back to its regular physiological condition. Moreover, there
is a likelihood that, providing ad-libitum access to feed after starvation led to excessive feed intake which
in turn stimulated unusually high secretion of pancreatin resulting in lower amylase activity in the
pancreas itself.
Effect of age. The effect of age on pancreatic amylase activities in birds (see section 2.5) and pigs was
studied by many. Laws and Moore (1963) observed interesting phenomenon in chicks, thus, high levels
of pancreatic amylase activity were found in the newly-hatched chick but these levels decreased during
the first 20 days after hatching and then remained constant. This finding with the chick is in contrast to
those reported for pigs. Pig amylase activity is low at birth but increases remarkably during the following
35 days (Laws and Moore, 1963). However, some studies (Lindemann et al., 1986, Owsley et al., 1986)
reported transient decrease in the activity of enzymes immediately after weaning and the total activity of
all enzymes increased with time post-weaning. Not much literature is known on the pancreatic enzyme
36
activity of older pigs. Kvanitski (1951) assessed enzyme activity of the pancreatic juice from 20 days to
200 days old pigs and found a decrease in proteolytic and lipolytic activity of the pancreatic juice
throughout increasing age, however amylase levels stayed relatively constant. Weström et.al (1987)
studied the development of pancreatic enzymes in pigs from 10 days up to 6 month of age. According to
their observations all enzyme activities increased over increasing age (as stated in (Makkink and
Verstegen, 1990). From these literature, it can be summarized that, enzyme activities for pigs and poultry
develops with increasing age of the animals with some fluctuations at certain stages of their lifespan.
Moreover, reports also show that feeding regimen, diet composition interacts with age dependency
(Makkink and Verstegen, 1990).
The pigs and the birds selected for our experiment were dissected at the same age (all pigs at 17 weeks of
age and all chicks at 21 days). Thus, individual variation in pancreatic amylase activity within species
cannot be explained by the effect of age. However, difference in the ages of the dissected pigs and chickens
emerges a question whether the chosen animals were suitable for comparative study or not. Chickens in
our experiment were killed at 3 weeks of age well before they reached sexual maturation at 5-6 month of
age. However, pigs were slaughtered at about 4 month of age which was fairly closer to maturity age (5-
6 month (Reiland, 1977)) compared to birds. Since, none of the animals have reached to maturity age we
cannot not expect dramatic shifts in the physiological status of the individuals. However, literature shows
that pancreatic amylase activity of the animals until sexual maturity can be irregular with age. Hence, it
is arguable whether pancreatic amylase activities of chickens and pigs in our experiment are representative
for the entire population of pigs and chickens.
Environmental conditions. The conditions which animals are kept can also change their pancreatic enzyme
output (Longland, 1991). Osman and Tanios (1983) postulated that pancreas plays an important role
during the adaptation of chickens to heat through the regulation of intestinal level of amylase. In the study,
broilers exposed to heat for 15 days increased biosynthesis of amylase in the pancreas, but not its secretion
to the intestine thus, levels of amylase in the intestine declined. Reverse relationship of amylase levels in
the pancreas and small intestine was monitored by the authors. However, in the same study heat
acclimatized laying hens lowered the amylase activity both in the intestine and pancreas. Previous
observation in broilers agree with the report by Routman et al. (2003) where they evaluated the effect of
temperature exposure (26 and 35ºC) and different diet energy levels (2,900 and 3,200 kcal ME/kg) on the
activity of digestive enzymes in broiler chickens. Based on their findings, pancreas amylase activity was
affected by energy level of the diet and by thermal stress. Heat stressed birds had a higher enzymatic
37
activity (p<0.05) than control birds. Furthermore, energy restricted birds also increased (p<0.01) their
pancreatic amylase compared to non-feed restricted group. Authors however, did not monitor the intestinal
enzyme activity.
In contrary, Szabo et.al (1976) found that piglets kept at high (38-40 ºC) temperature had lower lipase and
α-amylase levels in pancreatic homogenates than piglets kept at lower (0-2 ºC) temperature (Makkink and
Verstegen, 1990, Longland, 1991). Low enzyme activities in general can be explained as a physiological
adjustment to reduce body’s heat production by reducing the feed intake and consequently the synthesis
of the enzymes that are needed for digesting main energy sources such as lipids and carbohydrates.
However, Makkink and Verstegen (1990) claims that, in previously mentioned study feeding was ad-lib,
so that feed intake could have affected the results. Heat acclimatization does not seem very appropriate to
explain individual variation in pancreatic amylase activity within species in our experiment. Presumably
all animals from both species were kept under same normal ambient temperatures that would not disturb
the synthesis of pancreatic amylase and other enzymes.
5.3 Protein analysis
The Bradford protein assay is widely used assay to determine the concentration of protein in solution. The
assay is based on binding of Coomassie Brilliant Blue (CBB) G-250 dye to proteins which causes a change
in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm
which is monitored (Bradford, 1976). Binding probably involves hydrophobic interactions between the
anionic form of the dye and basic (Arg, His, Lys)/aromatic (Phe, Tyr, Trp) amino acid residues on the
protein (Compton and Jones, 1985).
Nevertheless, the assay is not free of disadvantages. The high dependence of the assay on protein
composition presents a major problem to the broad use of CBB binding as a quantitative protein assay. It
is, however, quite useful as a general, sensitive, semi-quantitative assay for proteins (Sapan et al., 1999).
It is generally believed that the assay monitors formation of a soluble blue complex (Marshall and
Williams, 1993). However, Marshall and Williams (1992) observed that insoluble proteins could also
contribute to the color formation during analysis. They demonstrated loss in color yield after
centrifugation and filtration of the initial assay mixture. This indicates that centrifugation speed and
duration has an important impact on removal of insoluble proteins and thus on accuracy of determination
of soluble proteins. Centrifugation speed and duration varies in the published literature. Several authors
38
used rather high centrifugation speed of the pancreatic homogenates prior to enzyme analysis
(30 000×g for 1 h (Jensen et al., 1998), 27,000 ×g for 10 min (Owsley et al., 1986), 14,000 x g for 30 min
(Routman et al., 2003)) and obtained sufficient results in enzyme activities. The speed was restricted to
4000 RPM ( ̴ 2700 ×g for 12 min) in our experiment which was the maximum speed of the centrifuge.
Thus, there is a possibility that analyzed samples might have contained insoluble proteins to a little extent
which would have interacted with color formation. This might have some consequences on accuracy of
protein quantification in general, however, it should not affect the comparability of the data.
α-amylase and other enzymes of pancreatic origin are considered as water-soluble enzymes (Schmid,
1979), except some derivatives of α-amylase enzymes (Ledingham and Hornby, 1969). Since pancreas
contains several enzymes and non-enzyme proteins, variation in the protein content can be due to varying
amounts of other enzymes (lipase, colipase, nuclease, proteases, etc.) assuming that concentration of non-
enzyme proteins in the pancreatic tissue is relatively constant.
5.4 Specific activity of α-amylase
Diet composition. Pancreatic amylase is very sensitive to any changes in the amount of starch intake
(Corring, 1980). Numerous studies have shown diet induced changes in the activity of several enzymes
of the pancreas in rats, pigs and birds. Desnuelle et al. (1962) and Abdeljlil et al. (1963) observed some
adaptations to a new diet in rats and stated that adaptation of the pancreas to dietary changes starts
immediately or almost immediately. Desnuelle et al. (1962) showed that specific amylase activity in the
rat pancreatic tissue increased six-fold when animals, adapted to a 20% starch diet, received food
containing 56 % of starch. Increasing the daily starch intake from 160 g to 600 g, caused 30 % average
increase of the specific amylase activity in pigs (Corring 1975 as cited in (Corring, 1980). Such responses
in the pigs can occur very rapidly with a change in the diet composition. Nunes and Corring (1979) found
that, when a high carbohydrate diet was fed instead of a high lipid diet pancreatic amylase increased
significantly within two hours of the diet change (Longland, 1991).
In their study, Abdeljlil et al. (1963) found statistically significant variations in the specific activities of
several enzymes of rat pancreas after one day in the change of the nutrient composition of the diet. As a
matter of fact, specific activities of the fresh pancreas homogenates were high for amylase when animals
received high-starch diet and similarly, chymotrypsinogen was higher when animals were given casein-
rich diet. Lipase behaved differently. It was low in the casein-rich diet and high on the others. The specific
lipase activity in pig pancreatic juice was 7 times higher when the daily amount of triglyceride intake
39
increased from 30 to 220 g, however adaptation of the lipase to the fat content of diet is much slower
compared to other pancreatic enzymes (Corring, 1980).
The effect of diet can be quite marked on both levels of digestive enzyme activities and on the amount of
digestive tract secretions (Longland, 1991). Level of trypsin appear to stay relatively irresponsive to diet
induced changes, except the presence of some anti-nutritional factors (trypsin inhibitors) in the diet.
Corring (1980) and Schumann et al. (1983) showed that the overall pancreatic secretion increased when
the diet contained untoasted soybean meal compared to toasted soybean meal. Zebrowska et al. (1983)
performed experiments with growing pigs (ca 35 kg) on two diets: diet A (purified): casein, wheat starch,
sucrose, soya oil and diet B (cereals): barley meal, soybean meal. With diet B they found higher pancreatic
juice secretion (2182 versus 1204 g per 24 hours), but no difference in enzyme activities (Makkink and
Verstegen, 1990). Similarly, Nitsan and Gertler (1972) reported that amylase was affected more than the
proteolytic enzymes by the quality of the diet, and responded more of the methionine supplementation of
the heated and raw soybean diet. The diet of both species contained soybean meal in our experiment. The
inclusion level of soybean meal in the diet of chickens was reasonable higher (23.3%) compared to pigs
(13%). Considering previously mentioned findings, it is also possible to imagine stimulation of amylase
or overall pacreatic secretions due to soybean meal in the diet of both species. However, it would be
difficult to speculate the magnitude of the influence for both type of animals.
Any alteration in the type or quantity of dietary proteins leads to an adjustment of specific and total
enzyme activities in the pancreatic tissue and pancreatic juice (Corring, 1980). This statement also
indicates that amylase activity in the pancreas is not dictated by the amount of starch substrate per se.
Presence of other protein polypeptides may also affect synthesis and secretion of amylase from the
pancreatic tissue. Corring et al. (1972) demonstrated that relative amounts of dietary protein and starch
may affect the degree of variation in the specific and total activities of amylase in the pig pancreatic tissue.
At very high levels of only starch intake (81 % starch, 0 % protein) specific amylase activity was not as
high as in the feeding regimen where diet contained considerable amount of protein (30% protein, 41 %
starch), although starch content was reduced by half. Similarly, the ratio of carbohydrate, protein and fat
in the diet could also influence lipase activity in pigs (Corring, 1980). Pekas et al. (1966) showed that
secretion of amylase, protease and lipase was about 5 times higher when heat treated soya-protein were
fed to 7 weeks old piglets compared to milk protein. This finding indicates again that not only the quantity
but also the quality of proteins has tremendous effect on enzyme activities of the pancreas.
40
Feed intake. Effect of feed intake on pancreatic enzymes activities was studied by León et al. (2014). In
the study, effect of starvation on pancreatic amylase activity was also monitored. Newly hatched broiler
chickens were assigned to three different dietary treatments from hatching to 48 hours: fasting, hydrated
balanced feed (HBF: 54% cornmeal, 35% soybean meal, 6% soybean oil) and commercial hydrating
supplement (CHS: soy flour 40%, corn flour 15%, corn syrup 18%, water 25%). After 48 hours, birds
were fed with commercial feed and specific enzyme activities (mg starch degraded/min/mg protein) were
measured immediately, 48 and 72 hours after hatching. The specific activity of pancreatic amylase peaked
to the highest level at 48 hours in the fasting birds which was about three times higher than the birds
receiving feed. However, a reduction in the amylase activity was detected 72 hours after hatching. On the
other hand, specific activity of lipase and trypsin followed the opposite pattern, remained relatively stable
up to 48 h and increased at 72 h in the fasting birds. In birds receiving HBF and CHS pancreatic enzyme
activity was reduced in the first 3 days of life of broiler chickens.
Palo et al. (1995) reported decrease in relative and specific activities of pancreatic amylase and protease
in the partially feed restricted birds compared to ad-libitum fed birds. Authors explained it as an adaptation
to the reduced feed intake and thus reduced substrate levels in the small intestine. Their observations were
in agreement with the report by Corring (1980) indicating that pancreatic amylase and protease secretions,
as well as their concentrations in pancreatic tissue were proportional to the amount of the substrate. These
responses also depend on the duration of the restriction period. Corring (1980) reviewed the literature on
the adaptation of digestive enzymes to the diet in pigs and indicated that when dietary restriction is not
too severe, the biosynthesis of all digestive enzymes markedly decreases (Palo et al., 1995).
Surprisingly, in spite of the fact that the diet, feeding, age, genetic stock, environmental conditions were
pretty standardized for birds and pigs, large variation was present in pancreatic amylase activities among
animals from both species. Although there is inconclusiveness in the literature in regard to the effect of
the feed intake and diet composition on pancreatic enzyme activities, it can be assumed that the large
variation in the pancreatic amylase activity is not very abnormal situation for the birds. Varying
concentration of amylase in chickens can primarily be the reason of varying extent of pancreatic emptying
caused by the effect of the feed restriction. Furthermore, ad-libitum feeding after feed restriction may have
resulted in varying amount of feed being consumed by the birds that would have consequently caused
dissimilar stimulation of synthesis and secretion of pancreatic enzymes.
Storage conditions. The measurement of amylase activity in the pancreas encounters several problems,
e.g., standardization of methods of analysis, enzyme activity changes in stored pancreatic juice and
41
interference by microbial contamination of the homogenates (Makkink and Verstegen, 1990). Some of
the challenges mentioned by Makkink and Verstegen (1990) were also experienced in the present study.
Although, enzyme assays were quite standardized for all pancreases throughout the experiment,
differences in storage conditions of the pancreases prior to enzyme activity analysis may as well impeach
the reliability of the comparative data. Effect of storage conditions on amylase activity was explicitly
discussed in the literature review (see section 2.6) and will be briefly reconsidered here.
Low (1982) analyzed digesta samples for trypsin, chymotrypsin and peptidases within a month of
collection and found that storage at – 200 C for less than 3 months did not influence enzyme activities in
the samples (Makkink and Verstegen, 1990). Legg and Spencer (1975) studied the stability of pancreatic
enzymes in human duodenal fluid to storage temperature and pH. They found that activity of amylase
remained relatively constant when samples were kept at – 200 C over a month period. At pH < 5 enzyme
activities substantially decreased within 4 weeks of storage at – 200C. Corring (personal communication,
1988) recommended storage of pancreatic juice at -80 °C to prevent loss of enzyme activities (Makkink
and Verstegen, 1990).
It is difficult to conclude any significant decrease in the amylase activity of the chicken pancreases based
on the reviewed literature. Since most of the studies evaluating enzyme activities of the samples stored at
-200 C restricted the duration of storage to less than 3 months.
42
6. CONCLUSION
The hypothesis that, pancreatic tissue of broiler chickens resulting in more efficient digestion of starch
was falsified in our experiment.
The in-vitro digestibility assay used in this experiment can be further improved in future studies. Thus,
amyloglucosidase and amylase digestion steps can be combined, digesta sample volumes can be reduced
and total reaction time can be extended.
More carefully controlled studies are needed in the future to study pancreatic amylase activity and
pancreases that are used must be standardized more on feeding and diet composition between species.
Storage conditions as well as assay methods should be standardized to facilitate comparison of the
research data.
43
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APPENDIX A
Table 1. Results of the starch digestibility analysis. Raw data for each assay round. Values represent glucose
concentration (mmol/L) of the digesta at different reaction times (min).
Sample
# Chicken 2 Pig 2
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.61 0.8 0.15 0.42 0.58
15 2.15 2.28 0.14 1.87 1.98
30 3.16 3.08 0.24 2.47 2.42
60 4.61 4.46 0.24 3.47 3.48
90 6.05 5.97 0.17 4.42 4.77
120 7.59 7.38 0.36 5.26 5.21
150 8.48 8.29 0.22 5.71 5.78
Sample
# Chicken 4 Pig 4
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.49 0.84 0.34 0.51 0.53
15 1.81 2 0.16 1.42 1.43
30 2.63 ------- 0.31 1.75 1.79
60 3.59 3.25 0.26 2.4 -------
90 4.27 ------- ------- 3.17 2.97
120 5.27 5.06 0.56 3.6 3.51
150 6.04 5.82 0.74 3.99 3.66
Sample
# Chicken 6 Pig 7
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.71 0.81 0.57 0.77 0.68
15 1.74 1.81 0.22 2.22 2.27
30 2.35 2.39 0.38 3.25 3.37
60 3.41 3.45 ------- 4.85 4.89
90 4.07 4.23 0.23 ------- 6.07
120 4.84 5.03 0.19 7.08 7.1
150 5.48 ------- 0.31 8.18 8.25
Sample
# Chicken 1 Pig 1
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.54 0.51 0.18 0.64 0.55
15 2.51 2.54 0.22 2.12 2.26
30 3.76 3.64 0.34 3.14 3.15
60 5.72 5.59 0.19 4.31 4.41
90 7.42 7.41 0.44 4.55 5.63
120 8.76 8.97 0.46 6.24 6.58
150 10.05 9.98 0.41 6.95 7.63
Sample
# Chicken 3 Pig 3
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.52 0.55 0.11 0.77 0.8
15 1.5 1.62 0.26 2.26 2.38
30 1.92 1.9 0.27 3.09 2.98
60 2.84 2.9 0.36 4.64 5.42
90 3.53 3.6 0.41 7.21 7.38
120 4.49 4.37 0.45 8.39 8.8
150 4.85 4.52 0.39 9.81 10.03
Sample
# Chicken 5 Pig 5
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.54 0.66 0.59 1.09 1.14
15 1.37 1.47 0.45 2.46 2.48
30 1.78 1.7 0.24 3.79 3.8
60 2.29 2.34 0.22 5.2 5.45
90 3.1 2.85 0.42 7.27 7.12
120 3.33 3.34 0.27 8.35 8.21
150 3.62 3.34 0.32 9.5 9.16
51
Sample
# Chicken 8 Pig 8
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.38 0.46 0 0.59 0.52
15 2.61 2.6 0.17 1.99 2.07
30 4.1 4.15 0.12 2.7 2.85
60 6.14 6.74 0.05 4.6 4.49
90 8.86 8.89 0.34 5.62 6.02
120 9.67 10.66 0.37 6.67 6.73
150 10.53 11.43 0.22 7.61 7.66
------- data was lost
Figure 1. Average blank values at different times. Values are shown as means + SD + CV%
Sample
# Chicken 7 Pig 7
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.52 0.58 0 0.34 0.44
15 2.11 2.12 0.19 1.9 1.7
30 3.37 3.34 0.32 2.47 2.66
60 5.29 5.02 0.13 3.66 3.71
90 7 7.07 0.23 4.79 4.73
120 8.62 8.59 0.35 5.73 5.66
150 9.77 9.44 0.19 6.01 6.22
Sample
# Chicken 9 Pig 9
Time Parallel
1
Parallel
2 Blank
Parallel
1
Parallel
2
0 0.51 0.38 0.15 0.61 0.69
15 1.21 1.34 0.23 1.51 1.37
30 1.54 1.51 0.1 2.02 2.07
60 2.23 2.37 0.2 3.02 3.16
90 2.95 2.76 0.27 3.94 3.87
120 3.22 3.24 0.12 4.29 4.2
150 3.49 3.49 0.23 5.01 5.01
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0 15 30 60 90 120 150
Glu
cose
(m
mo
l/L
)
Time (min)
95 %
41 %
37 %
44 %
33 %
40 % 51 %
52
Table 2. Body weights (BW) and pancreas weights (PW) of the two species.
Pig Chicken
# BW ˣ103 (g) PW (g) BW* (g) PW (g)
1 108.5 128 Zeolite 1107
2.1
2 108.9 139 2.8
3 109.4 125 2.7
4 111.1 137
Marble 996
2.7
5 114.5 141 2.3
6 115.5 126 2.5
7 107.3 129 2.8
8 115.9 104 Granite 1070
2.2
9 112.1 137 2.1
Average 111.5 130 1050 2.4
SD 3.2 11 53 0 * Average body weights of the chickens for different treatment groups
53
APPENDIX B
Figure 1. Standard curves with H2O (--●--) and Tris-HCl buffer (––▲––)
Figure 2. BSA standard curve. Output from the software (SoftMax).
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20 25
Ab
sorb
ance
at
59
5 n
m
Protein concentration (mg/ml)
54
Table 1. Results of the protein analysis. Mean results are given as μg/ml unit of protein.
Sample Well
Values
(absorbance
at 595nm)
Result MeanResult Std.Dev. CV%
1
A3 0.145 9.277
9.248 0.258 2.789 A4 0.14 8.977
A5 0.149 9.491
2
B3 0.119 7.771
7.506 0.255 3.393 B4 0.114 7.483
B5 0.11 7.264
3
C3 0.145 9.266
9.164 0.182 1.981 C4 0.145 9.271
C5 0.14 8.954
4
D3 0.127 8.21
8.137 0.262 3.215 D4 0.129 8.354
D5 0.12 7.846
5
E3 0.136 8.752
8.64 0.099 1.149 E4 0.134 8.608
E5 0.133 8.562
6
F3 0.114 7.471
7.44 0.107 1.441 F4 0.111 7.321
F5 0.115 7.529
7
G3 0.123 7.979
7.89 0.195 2.466 G4 0.124 8.025
G5 0.117 7.667
8
H3 0.114 7.5
7.815 0.665 8.503 H4 0.112 7.367
H5 0.133 8.579
9
A6 0.127 8.198
8.212 0.084 1.029 A7 0.128 8.302
A8 0.126 8.135
10
B6 0.143 9.139
8.9 0.229 2.569 B7 0.138 8.879
B8 0.135 8.683
11
C6 0.111 7.298
7.052 0.286 4.053 C7 0.101 6.739
C8 0.108 7.119
12
D6 0.118 7.731
7.617 0.497 6.529 D7 0.124 8.048
D8 0.107 7.073
13
E6 0.067 4.742
4.592 0.145 3.148 E7 0.064 4.581
E8 0.062 4.454
55
14
F6 0.132 8.498
8.564 0.066 0.775 F7 0.134 8.631
F8 0.133 8.562
15
G6 0.121 7.875
7.687 0.165 2.145 G7 0.116 7.569
G8 0.116 7.615
16
H6 0.168 10.592
10.302 0.253 2.46 H7 0.16 10.125
H8 0.161 10.189
16a4
C9 0.13 8.412
8.685 0.256 2.948 C10 0.136 8.723
C11 0.139 8.919
17
A9 0.057 4.194
3.977 0.211 5.303 A10 0.053 3.963
A11 0.05 3.773
18
B9 0.133 8.55
8.535 0.059 0.694 B10 0.133 8.585
B11 0.131 8.469
4 Sample was diluted more (1:3750) to fit to the standard curve
56
APPENDIX C
Table 1. Results of the simple linear regression analyses (output from SPSS). Relationship between starch
digestibility and protein content of the pancreas P > 0.05. (Species 1= pig, Species 2=chicken)
Model Summaryb,c
Model
R
R Square
Adjusted R
Square
Std. Error of the
Estimate
Species =1.00
(Selected)
Species ~= 1.00
(Unselected)
1 .402a . .161 .042 17.89733
a. Predictors: (Constant), Protein pancreas b. Unless noted otherwise, statistics are based only on cases for which Species =1.00. c. Dependent Variable: Starch digestibility
Model Summaryb,c
Model
R
R Square
Adjusted R
Square
Std. Error of the
Estimate
Species =2.00
(Selected)
Species ~= 2.00
(Unselected)
1 .131a . .017 -.123 27.91072
a. Predictors: (Constant), Protein pancreas b. Unless noted otherwise, statistics are based only on cases for which Species =2.00. c. Dependent Variable: Starch digestibility
Table 2. General linear model (GLM) (output from SPSS). Starch digestibility as a dependent variable, time and
species as fixed factors.
Dependent Variable: Starch digestibility
Source
Type III Sum of
Squares df Mean Square F Sig.
Corrected Model 46464.930a 7 6637.847 35.287 .000
Intercept 142542.776 1 142542.776 757.755 .000
Time 46457.219 6 7742.870 41.161 .000
Species 7.711 1 7.711 .041 .840
Error 22197.218 118 188.112
Total 211204.924 126
Corrected Total 68662.149 125
a. R Squared = .677 (Adjusted R Squared = .658)
57
Table 3. Independent sample t test (output from SPSS) to compare the means of relative pancreas weights of pigs
and broiler chickens. (Species 1= pig, Species 2=chicken)
Group Statistics
Species N Mean Std. Deviation Std. Error Mean
Relative pancreas weights 1.00 9 .1156 .01130 .00377
2.00 9 .2356 .03087 .01029
Independent Samples Test
Levene's Test for
Equality of
Variances t-test for Equality of Means
F Sig. t df
Sig. (2-
tailed)
Mean
Difference
Std. Error
Difference
99% Confidence
Interval of the
Difference
Lower Upper
Relative
pancreas
weights
Equal variances
assumed 8.286 .011 -10.952 16 .000 -.12000 .01096 -.15200 -.08800
Equal variances
not assumed -10.952 10.108 .000 -.12000 .01096 -.15464 -.08536
58